Esters of penicillin from active halogen compounds



Patented Aug. 25 1953 UNITED STATES PATENT OFFICE 2,650,218 ESTERS OFPENIOILLIN FROM ACTIVE HALOGEN CO Douglas E. Cooper, Sy to BristolLaboratories N. Y., a corporation of MPOUNDS Oak Ridge, Tenn, and mouse,.N. Y., .assignors Inc., Onondaga County, New York No Drawing.Application April 11, 1952,

Serial No.

3 Claims. (Cl. 260--239.1)

This invention relates to esters of penicillin and to methods for theirproduction through the reaction of penicillin salts "with certainsubstituted alkyl halides.

The compounds of the present invention have the following generalformula:

ren-oo- JI X where Pen-COO represents a penicillin with the hydrogenatom removed from the carboxyl group, X represents a radical whichcontains, at the point of its attachment to the carbon atom, a CO-,--CN, or O-1inkage. Y represents hydrogen, an aryl group and X.

The structure of penicillin is now well known to the art. For purposesof this invention, it is considered to include compounds having thecharacteristic bi-cyclic ring system of .afi-lactam fused to athiazolidine carboxylic acid, and showing antibiotic activity.

Heretofore, a small number of the simpler esters of penicillin have beenobtained by use of the reaction of penicillin with diazomethane and itshomologs or derivatives. Such esters include methyl, ethyl, butyl,benzyl, and benzohydryl. None of these has shown any therapeuticactivity in man, however, since these esters are substantially inactivefor the reason that hydrolysis to penicillin in the body proceeds tooslowly to be Thesecompounds are all characterized by high reactivitytoward nucleophilic reagents in the second order displacement reaction.Organic reaction theory indicates that the carbon atom bearing thehalogen has been activated by the attached group and, as a result, bearsa small increment of positive charge which facilitates the access of thenucleophi-lic reagent (base) and thus lowers the activation energy ofthe reaction.

The alkyl halides which the penicillin esters of this invention fallinto several sub-classes:

Sub'C1ass Illustrative Compound 1. 2-oxo-alkyl halides Cl'CHz-COCH3 6.l-alkoxy alkyllialides C1CHzO-O.H3

7. l-aryloxy alkyl halides C1CH-OO,;H5

It will be understood that other halides (bromides and iodides) may beused in place of the alkyl chlorides listed above. Becauseof the hazardssurrounding the use of fluorine compounds, it has been foundadvantageous to use one of the other halides. Also other penicillinsalts (calcium, potassiumammonium, etc.).may be used instead of thesodium salt.

The following examples will serve .to illustrate the invention:

Example 1.-Pem'czllz'n acetom Z ester: Pen COONa+ClCHzCOCHa-Pen-COO-CH2COCH3 C-hloroacetone (4.7 ml.) was added to a cold swirlingsolution of g. sodium penicillin in .water) and the solution stored inthe refrigerator. On the next day the white crystals were filtered off,washed with 25% isopropanol and dried; yield 10.97 g. (50%); M. P.92 Forassay 10.2 mg. was dissolved in 20 m1. isopropanol and diluted to 100ml. with neutral buffer; iodometric 1300 units/mg, S. aureus 1430 unitsper mg. (theory=1520). Because aqueous solutions of this ester undergohydrolysis at a substantial rate, it is not determined what proportionof the activity shown against S. aureus may be due to unhydrolyzed esteras compared with free penicillin released during the incubation of theassay plate.

Analysis:

Found Calculated Example 2.-Pem'cillin phenacyl ester:

Pen 000m GoHsOOOHABI HO ONMc Pen O O CH2C O C0115 A nearly pure grade ofsodium penicillin G (100 g.) was dissolved in 500 ml. dimethylformamide.Addition of 56 g. of phenacyl bromide released considerable warmth.After storage in the refrigerator overnight, the solution was pouredinto a mixture of 500 ml. benzene and 2500 ml. neutral buffer. Thebenzene layer was separated and combined with another 500 ml. extract ofthe aqueous layer, then dried over sodium sulfate. Skellysolve D wasthen added to the point of incipient turbidity, and the solution was letstand one hour. An excellent crop of white crystals, weighingapproximately 60 g. separated. A second addition of Skellysolve D to thecloud point produced another crop of about the same amount, but slightlytan in color. The third addition gave a white product again but insmaller quantity. Still slightly wet with benzene, the combined cropswere dissolved in 250 ml. acetone; the solution was diluted withSkellysolve D and stored in the refrigerator overnight. The colorlessneedles which separated were washed with a 1:2 mixture of acetone andSkellysolve D the solution was diluted with Skellysolve D, and dried;yield 4.4 g.; M. P. 136-1365".

Analysis:

Calculated Found For assay 8.52 mg. was dissolved in 35 ml. absoluteethanol and the solution diluted to 100 ml. with neutral buffer;iodometric 1362 units/mg. (theory=1320) S. aureus 221 units/mg. As withthe acetonyl ester and other esters of this group the facility ofhydrolysis leaves it uncertain The solid was filtered, washed with(though unimportant) what preparation of the activity against bacteriais due to intact ester.

Example 3.-Penicillin p-bromophenacyl ester:

Pen COONa+BrC COCHzBr MeCONMea Pen 00001120 0C Br+NaBr A cold solutionof 1 g. sodium penicillin G in 10 ml. acetyldimethylamine was treatedwith 0.65 g. p-bromophenacyl bromide, and the solution kept in therefrigerator overnight. Dilution with ml. neutral phosphate bufierproduced a solid white precipitate which was filtered off, washed withwater, then dried in vacuo; yield 0.57 g. (46%). For assay 50 ml. wasdissolved in ethanol and the solution diluted with a small amount ofneutral buffer; Iodometric assay: 1050 units/mg. (theory=1100). It wasrecrystallized from a supersaturated soltuion prepared by adding waterto an isopropanol solution to the point of incipient turbidity.

Analysis: Calculated for C24H23N205B1.

\ Calculated Found Example 4.-p-Phenylphenacyl ester:

To a chilled solution of 1 g. sodium penicillin in 10 ml.acetyldimethylamine was added 0.55 g. p-phenylphenacyl bromide, and thesolution was stored overnight in the refrigerator. Addition of thereaction mixture to 10 volumes of buffer precipitated the ester as aWhite solid. It was filtered off, washed with water, then dried invacuo. The material later became gummy, but when rubbed with isopropylalcohol the mass disintegrated into white crystals. The dry solidweighed 0.64 g. (M. P. l05-113) This was dissolved in 8 ml. benzene, andthe solution centrifuged to separate inorganic impurities. Addition of 6ml. cyclohexane produced a faint turbidity in the solution, from whichcolorless crystals separated when it was stored in the cold overnight;yield 0.43 g. (67%) M. P. 120.5-22". an accurate iodometric assay onthis compound because of its insolubility even in highly alcoholicaqueous solutions. The best value was 546 units/mg. (theory =1120). TheS. aureus assay showed 37.6 units/mg, indicating either a very slow rateof hydrolysis, or destruction by the necessarily high alcoholic contentof the solution.

Analysis:

\ Calculated Example 5.-Penicillin carboramz'domethyl ester:

Pen COONa OICHzCONHz HCONMez Pen 00 O OH2CONHI NaCl It was difficult toobtain was allowed to stand two days at room temperature. The end of thereaction is marked by clearing. of the liquid suspended. sodiumchloride, which finally forms a White: layer in the flask bottom. Enoughof the solution to produce a faint turbidity was added to 2000 ml.buffer and seed crystals, obtained by rubbing the oil produced in asimilar but smaller scale experiment, were introduced. Slow addition ofthe remainder of the dimethylformamide solution with stirring was thenresumed. After addition was complete stirring was continued five minuteslonger; the crystals were filtered ofi and washed with buffer thenwater; yield 12.9 g. (29%). For assay 9.46 mg. was dissolved in 7 ml.isopropanol and the solution diluted to. 50. ml. with buffer; iodometric1450 units/mg. (theory=1520). Solution of 12.8 ml. in the 400 ml.acetone, followed by dilution to the turbid point with Skellysolve D(500 ml.) produced on standing 9.55 g. crystals; M. P. 165-7 (dec.).This compound is soluble inmost organic solvents including benzene, butit is only slightly soluble in Water.

Analysis: Calculated for C18H21N306S Calculated Found Ewample:6.Pem'cillin carbethoxymethyl ester:

Pen OOONa CHgBrOOOCzIL HCONMe; Pen COOCHzCOO 02H; NaBr day, the whitecrystalline solid was filtered off, 4

washed with buffer, then water, and dried in vacuo; yield, 14.1 g.(80%). For recrystallization, 14 g. was dissolved in 350 ml. ether andthe solution filtered from inorganic impurities. Addition of 120 m1. ofSkellysolve turbidity; the solution was seeded and allowed to stand atroom temperature for half an hour, then in the refrigerator for anotherhour. The crystals, washed with Skellysolve A and dried in the air,weighed 6.0 g.; M. P. 76-775". A second Skellysolve D addition followedby standing overnight gave a crop which was slightly yellow; 3.28 g.Recrystallized from ether and Skellysolve, 1.63 g.; M. P. 77-775. Thetotal, 7.63 g., represents a 43% yield of purified ester. For assay12.73 mg. was dissolved in 5 ml. ethanol and diluted to 100 ml. withneutral buffer; iodometric, 1210 units/mg. Theory=1410; S. aureus 43units/mg, ratio subtilis/staph., 0.39. The latter figure is sufficientlyshifted from the 1.0 value to be expected of sodium penicillin G toindicate at least a part of the bacteriostatic activity was due to theester per se.

Analysis: Calculated for CH24N206s D produced a faint r Example7.--PeniciZZin1 methoxymethyl ester: Hen. COONa+ClCH2OCHa- PenCOOCI-IzOCHa A cold solution containing 1 g., sodium penicillin G- in 10ml. acetyldimethylamine was treated with- 0.3 ml. chloromethyl. etherand allowed to stand in. the refrigerator overnight. Addition of thereaction. solution. to. ml. bufl'er produced a gummy precipitate which.wa extracted with 50 m1. chloroform. The chloroform solution was washedwith, 25 ml. neutral bufier and then. 25 ml. water to free it of anyunreacted penicillin. Assays on 230 ml. aqueous solution prepared from 1ml. of the chloroform solution showed 681,000 units iodometrically and.510,000 units. on S; au'reus plates. Evaporation of the chloroformsolution gave a slightly yellow oil, which was dried; H1311. highvacuum; yield 0.47 g. (46%)..

To make certain of. the absence of free penicillin it was againdissolved in 50 ml. chloroform, washed. with 50 m1. neutral buffer,then. 50 ml. water. An. aqueous solution (30 ml.) prepared from 1 m1. ofthe chloroform solution showed the total. solution. still to contain190,000 units, corresponding; to. 405 units/mg. for the ester(theory=1760)'.

A.- suitable solvent for the reaction. by which the esters of thisinvention are formed must, in addition to its solvent qualities, beindifferent toward penicillin, the alkyl halide used in the reaction,and the ester formed by the reaction, all of which are relativelyreactive compounds. In addition to possessing these qualities, it isadvantageous that the solvent be miscible with water in order tofacilitate isolation and purification of the product.Acetyldimethylamine and dimethylformamide have been found especiallysuitable. The reaction of a penicillin salt with the alkyl halideproceeds smoothly in these solvents and produces high yields of thedesired esters. These can readily be isolated as crystalline solids bysimple dilution with buffer or water, in which the majority of thedesired ester are almost insoluble. The simplicity and economy, and alsothe wide applicability, of these operations present strong contrastswith the conditions and limitations of the diazomethane method used forthe preparation of the penicillin esters previously known.

The esters of the present invention contain groupings which labilize theester linkage, and, in contact with blood or other aqueous fluids, theyrelease active penicillin at marked rates saponification. Furthermore,these rates are subject to very wide variation in different esters. Asan example of this, the relative rates of hydrolysis or saponificationof several such esters in a buffered aqueous acetone solution were foundto be as follows:

These rates fall within the range desired for therapeutic usefulness.Thus, at the pH of blood (approximately 7.35) and at body temperature(37 0.), the rate of saponification of the acetonyl ester corresponds to50% hydrolysis (to active penicillin) in 75 minutes.

One of the characteristics of esters or the Present invention whichcontributes to their usefulness is their low rate of solution in aqueousfluids. This property, like the rate of hydrolysis, is also variable,and, by selection of members having the proper rates of solution andhydrolysis, therapeutically valuable repository products may beobtained. Thus, upon intramuscular injection of an aqueous suspension ofsuch an ester, a process of slow solution and hydrolysis by body fluidsis initiated, resulting in the prolonged maintenance of therapeuticconcentrations of penicillin in the blood.

Another property of many of the new esters which is important for oralpenicillin preparations is their freedom from the bitter taste whichcharacterizes penicillin salt.

7 Still another useful property of the new esters is their resistance tothe action of penicillinase, an enzyme highly destructive to penicillinand present in a substantial proportion of wound and other infections aswell as in the normal intestinal tract.

In addition to the above useful properties of the penicillin esters ofthis invention, these esters also provide a means for protecting thecarboxyl group of penicillin through a series of chemicaltransformations, and for allowing sub-- sequent regeneration of thecarboxyl groupupon hydrolysis. Hence. these compounds are Valuable asintermediates. 1 x

We claim: t' l. A new class of compounds of the general formula PenCOOCH2CO-R H r where R is a member selected from the group consisting ofamino, (lower) alkylamino, di-, (lower) alkylamino, and (lower) alkoxyand where Pen COO represents a penicillin with the hydrogen atom removedfrom the carboxyl roup.

.2. Penicillin carboxamidomethyl ester.

3. Penicillin carbethoxymethyl ester.

HAROLD F. McDUFFIE, JR[ 1 DOUGLAS E. COOPER.

References Cited in the fileof this patent UNITED STATES PATENTS NameDate McDuflie et al Dec. 11, 1951 OTHER REFERENCES Carpenter J. Am.Chem. Soc. vol. 70, Sept. 1948, p. 2966.

Number

1. A NEW CLASS OF COMPOUNDS OF THE GENERAL FORMULA